Sexing method of bovine embryos

Abstract

The present invention provides PCR primers with which sexing of bovine embryos can be easily attained and provides a practical, rapid and reliable method for determining the sex of bovine embryos using these primers. The methods for determining the sex of the bovine embryos are characterized by discriminating PCR products which are obtained by amplifying specific DNA sequences by PCR with pairs of male-specific and gender-neutral primers. These primers are derived from DNAs which specifically hybridize to the bovine male genome and from DNAs which gender-neutrally hybridize to both bovine male and female genomes.

We claim: 
     
       1. A method for sexing bovine embryos, comprising conducting a PCR reaction wherein a small part of a bovine embryo provides the DNA template for the PCR reaction and wherein a first and a second pair of isolated single stranded DNA molecules are used for the PCR; wherein said first pair of DNA molecules consists of 10-40 contiguous nucleotides from a DNA molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 and said second pair of DNA molecules consists of 10-40 contiguous nucleotides from a DNA molecule selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; subjecting the resultant amplified PCR products to gel electrophoresis; and analyzing the gel electrophoresis. 
     
     
       2. An isolated DNA molecule as shown in SEQ ID NO:4 which comprises two nucleic acid sequences, of which one nucleic acid sequence hybridizes specifically to bovine male genomic DNA and the other nucleic acid sequence hybridizes to both male and female bovine DNA. 
     
     
       3. A method of sexing bovine embryos according to claim 1 in which the first pair of isolated single stranded DNA molecules consists of isolated single-stranded DNA molecules which specifically hybridize to bovine male DNA wherein the DNA molecules consist of 10 to 40 contiguous nucleotides from a DNA molecule represented by the DNA sequence SEQ ID NO:2. 
     
     
       4. An isolated DNA molecule which hybridizes to both bovine male and female genomic DNA, wherein the isolated DNA molecule is represented by a DNA sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 or a complementary strand thereof. 
     
     
       5. An isolated single stranded DNA molecule which specifically hybridizes to bovine male DNA, wherein the DNA molecule is represented by a DNA sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12 and SEQ ID NO:13, or a complementary strand thereof. 
     
     
       6. A method of sexing bovine embryos according to claim 1 in which the first pair of isolated single stranded DNA molecules consists of isolated single stranded DNA molecules which specifically hybridize to bovine male DNA, wherein the DNA molecules consist of 10 to 40 contiguous nucleotides from a DNA molecule represented by the DNA sequence of SEQ ID NO:3. 
     
     
       7. A method of sexing bovine embryos according to claim 1 in which the first pair of isolated single stranded DNA molecules consists of the isolated single stranded DNA molecules which specifically hybridize to bovine male DNA, wherein the DNA molecules consist of 15 to 25 contiguous nucleotides from a DNA molecule represented by the DNA sequence of SEQ ID NO:3. 
     
     
       8. The DNA molecule according to claim 4 which is represented by the DNA sequence, SEQ ID NO:5. 
     
     
       9. The DNA molecule according to claim 4 which is represented by the DNA sequence, SEQ ID NO:6. 
     
     
       10. The DNA molecule according to claim 4 which is represented by the DNA sequence, SEQ ID NO:7. 
     
     
       11. A method of sexing bovine embryos according to claim 1 in which the second pair of isolated single stranded DNA molecules consists of isolated single-stranded DNA molecules capable of hybridizing to both bovine male and female genomic DNA, wherein the DNA molecules consist of 10-40 contiguous nucleotides from DNA molecules selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 or a complementary strand thereof. 
     
     
       12. A method of sexing bovine embryos according to claim 1 in which the second pair of isolated single stranded DNA molecules consists of isolated single-stranded DNA molecules capable of hybridizing to both bovine male and female genomic DNA, wherein the DNA molecules consist of 15-25 contiguous nucleotides from DNA molecules selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO7 or a complementary strand thereof. 
     
     
       13. An isolated single stranded DNA molecule which hybridizes to both bovine male and female genomic DNA, wherein the DNA molecule is represented by a DNA sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, or a complementary strand thereof. 
     
     
       14. An isolated single stranded DNA molecule which hybridizes to both bovine male and female genomic DNA, wherein the DNA molecule is represented by a DNA sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, or a complementary strand thereof. 
     
     
       15. An isolated single stranded DNA molecule which hybridizes to both bovine male and female genomic DNA, wherein the DNA molecule is represented by a DNA sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:11. 
     
     
       16. A method for sexing bovine embryos, comprising conducting a PCR reaction wherein a small part of an embryo provides DNA templates for the PCR and first and a second pair of isolated single stranded DNA molecules are used for the PCR; wherein (i) said first pair of DNA molecules consists of DNA molecules selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:13, SEQ ID NO:19, a combination of SEQ ID NO:9 and SEQ ID NO:13, a combination of SEQ ID NO:8 and SEQ ID NO:13, a combination of SEQ ID NO:8 and SEQ ID NO:9, a combination of SEQ ID NO:14 and SEQ ID NO:15, a combination of SEQ ID NO:16 and SEQ ID NO:17, and a combination of SEQ ID NO:18 and SEQ ID NO:19; and   (ii) said second pair of DNA molecules consists of DNA molecules selected from the group consisting of SEQ ID NO:10, SE ID NO:11, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, a combination of SEQ ID NO:10 and SEQ ID NO:11, a combination of SEQ ID NO:20 and SEQ ID NO:21, a combination of SEQ ID NO:22 and SEQ ID NO:23, a combination of SEQ ID NO:24 and SEQ ID NO:25, and a combination of SEQ ID NO:26 and SEQ ID NO:27; subjecting the resultant amplified PCR products to gel electrophoresis; and analyzing the gel electrophoresis.